Damage due to insects costs billions of dollars annually in form of crop losses and in the expense of keeping these pests under control. The losses caused by pests in agricultural production environments include decrease in crop yield, poor crop quality, increased harvesting costs, and loss to health and environment.
Reference may be made to Hofte H. and Whiteley H. R., 1989, “Insecticidal crystal protein of Bacillus thuringiensis”, Microbiol. Rev. 53: 242-255, wherein Bacillus thuringiensis (B.t.) is a ubiquitous gram-positive spore-forming soil bacterium, known for its ability to produce parasporal crystalline inclusions during sporulation. These inclusions consist of proteins known as crystal proteins or Cry proteins or δ-endotoxins, which exhibit insecticidal activity, particularly against larvae of insect species in orders lepidoptera, diptera and coleoptera. Proteins with toxicity to insects of orders hymenoptera, homoptera, orthoptera, mallophaga; nematodes; mites and protozoans have also been mentioned in literature (Feitelson J. S., 1993, “The Bacillus thuringiensis family tree”, 63-71, In L. kim ed. Advanced engineered pesticides. Marcel Dekker. Inc., New York., N.Y. and Feitelson et al., 1992, “Bacillus thuringiensis: insects and beyond”, Bio/Tech. 10: 271-275; may be sited for this). Several strains of Bacillus thuringiensis (B.t.) have been identified with different host spectra and classified into different subspecies or serotypes on the basis of flagellar antigens. Pasteur Institute, France has catalogued 55 different flagellar serotypes and 8 non-flagellated biotypes. The reference may be made to Schnepf et al., 1998, “Bacillus thuringiensis and its pesticidal crystal proteins”, Microbiol. Mol. Biol. Riv. 62: 775-806, wherein several B.t. toxin-coding genes have been cloned, sequenced, characterized and recombinant DNA-based products have been produced and approved for commercial use. Through the employment of genetic engineering techniques, new approaches have been developed for delivering these B.t. toxins to agricultural environments, including the use of the genetically engineered crops and the stabilised intact microbial cells as δ-endotoxin delivery vehicles (Gaertner, F. H., Kim L., 1988, TIBTECH 6: 54-57). Thus, δ-endotoxin genes coding for proteins targeted to kill hosts, especially pests and insects that cause economic losses are becoming commercially valuable.
Commercial use of B.t. pesticides in a given crop environment is limited because a given δ-endotoxin shows toxicity to a narrow range of target pests. Preparations of the spores and crystals of B. thuringiensis subsp. kurstaki have been used for many years as commercial insecticides against lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces several δ-endotoxins, and is therefore toxic to a relatively broader range of lepidopteran insects. However, formulations based on the known δ-endotoxins, including B.t.k. HD-1 are not effective against some of the important crop pests, like Spodoptera sp. that also belong to order lepidoptera. Other species of B.t., namely israelensis and tenebrionis have been used commercially to control certain insects of the orders diptera and coleoptera, respectively (Gaertner, F. H., 1989, “Cellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms,” in Controlled Delivery of Crop Protection Agents, R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255; Couch T. L., 1980, “Mosquito Pathogenicity of Bacillus thuringiensis var. israelensis”, Development in Industrial Microbiology 22: 61-76 and Beegle C. C., 1978, “Use of Entomogenous Bacteria in Agroecosystems”, “Developments in Industrial Microbiology 20: 97-104; may be sited for this). Kreig et. al. (1983) in Z. ang. Ent. 96: 500-508, describe Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera i.e., Colorado potato beetles, Leptinotarsa decemlineata and Agelastica alni. 
Reference may be made to Crickmore et. al., 1998, “Revision in the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins”, Microbiol. Mol. Biol. Rev. 62: 807-813, wherein crystal protein genes are classified into 22 classes, primarily on the basis of amino acid sequence homology. The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in several cases in the published literature (Schnepf et al., 1981, “Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli” may be cited for this). U.S. Pat. Nos. 4,448,885 and 4,467,036 disclose the expression of B.t. crystal protein in E. coli. U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose B. thuringiensis strain tenebrionis, which can be used to control coleopteran pests in various environments. U.S. Pat. No. 4,918,006 discloses B.t. toxins having activity against dipterans. U.S. Pat. No. 4,849,217 discloses B.t. isolates, which have activity against the alfalfa weevil. U.S. Pat. No. 5,208,017 discloses coelopteran-active Bacillus thuringiensis isolates. U.S. Pat. Nos. 5,151,363 and 4,948,734 disclose certain isolates of B.t., which have activity against nematodes. Extensive research and resources are being spent to discover new B.t. isolates and their uses. As of now, the discovery of new B.t. isolates and new uses of the known B.t. isolates remains an empirical, unpredictable art. Several laboratories all over the world are trying to isolate new δ-endotoxin genes from B. thuringiensis for different host range and mechanism of action.
Bulla et al., 1980, “Ultrastructure, physiology and biochemistry of Bacillus thuringiensis”. CRC Crit. Rev. Microbiol. 8: 147-204 and Grochulski et al., 1995, “Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation”, J. Mol. Biol, 254: 447-464; have reported that majority of B.t. insecticidal crystal proteins are synthesised in natural form as protoxins (molecular weight 130-140 kDa), which form parasporal inclusions by virtue of hydrophobic interactions, hydrogen bondings and disulfide bridges. The protoxins, which are not toxic to insect larvae, are composed of two segments the—N-terminal half and C-terminal half. The protoxins are converted into functionally active toxins (60-70 kDa) in insect mid gut following their site-specific cleavage by proteases at alkaline pH. Such proteolytically processed, truncated δ-endotoxins bind to specific receptors in insect mid-gut and cause mortality by making pores in the epithelial membrane (the references, Bietlot et al., 1989, “Facile preparation and characterization of the toxin from Bacillus thuringensis var. kurstaki”, Biochem. J., 260: 87-91; Choma et al., 1990, “Unusual proteolysis of the protoxin and toxin from Bacillus thuringensis: structural implications”, Eur. J. Biochem. 189: 523-27; and Hofte et al., 1986, “Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringensis berliner 1715”, Eur. J. Biochem. 161: 273-280; may be cited for this). The protease-resistant active toxin corresponds to N-terminal half of the protoxin molecule. The other segment corresponding to C-terminal half is believed to be required for the formation of highly stable crystals. During the proteolytic processing, a small polypeptide comprising about 25-30 amino acid residues is removed from N-terminal of the protoxin.
The crystal structure of the core toxic segment of Cry1Aa and Cry3Aa δ-endotoxins are known (Grochulsky et al., 1995, “Bacillus thuringensis CryIA(a) insecticidal toxin: crystal structure and channel formation”, J. Mol. Biol. 254: 447-464 and Li et al., 1991, “Crystal structure of insecticidal δ-endotoxin from Bacillus thuringiensis at 2.5 Å resolution”, Nature. 353: 815-821) and their three-dimensional structures are superimposable. Reasonably conserved polypeptide domains suggest that related toxins have similar topological structure. These are globular molecules composed of 3 distinct structural domains connected by small peptide linkers. There are no crossovers of the polypeptide chains between the domains. Domain I consists of 7 α helical structures. Domain II consists of three anti-parallel β-sheets and two short α-helices. Domain III is a β-sandwich of two anti-parallel highly twisted β-sheets. Domains II and III are located on the side where they face helix α 7 of domain I. These domains are closely packed by virtue of numerous van der Wall forces, hydrogen bonds and electrostatic interactions (salt bridges) between the domains.
One of the major reasons for narrow host range of δ-endotoxins is that these proteins need specific receptors in the insect gut in order to make pores and cause toxicity. Since a given δ-endotoxin exhibits toxicity to a very narrow range of insects, it is desirable to engineer these proteins for modifying their receptor recognition in larval midgut, to widen host range and to improve toxicity. Two approaches have been followed for this purpose—first, the development of chimeric (or hybrid) genes by exchanging functional domains of the proteins and secondly, the development of improved δ-endotoxin proteins by site directed mutagenesis. References may be made to U.S. Pat. Nos. 5,128,130 and 5,055,294 wherein hybrid B.t. crystal proteins have been constructed, which exhibit increased toxicity and display an expanded host range to the target pests.
The reference may be made to Honee et al., 1990, “A translation fusion product of two different insecticidal crystal protein gene of Bacillus thuringensis exhibits an enlarged insecticidal spectrum” Appl. Environ. Microbiol. 56: 823-825, wherein translational fusion of two cry genes (cry1Ab and cry1Ca) has been made. The resulting hybrid protein had wider toxicity spectrum that overlapped those of the two contributing parental crystal proteins. However, the drawback is that the activity of the chimeric toxin did not increased over any of the parental toxins towards the target insect pests. In spite of poor toxicity, fusion gene was expressed in tobacco after partial modification, which conferred only partial protection to transgenic plants against a broader range of insects, including Spodoptera exigua, Heliothis virescens and Manduca sexta (the reference, van der Salm et al., 1994, “Insect resistance of transgenic plants that express modified Bacillus thuringensis cryIAb and cryIC genes: a resistance management strategy”, Plant Mol. Biol. 26: 51-59, may be cited for this).
The reference may be made to Honee et al., 1991, “The C-terminal domain of the toxic fragment of Bacillus thuringensis crystal protein determines receptor binding”, Mol. Microbiol. 5: 2799-2806, wherein 11 chimeric genes have been constructed using cry1Ab and cry1Ca as parent genes by exchanging functional domains. The draw back is that only two chimeric proteins, in which pore-forming domains had been exchanged, exhibited insecticidal activity. However, the efficacy of the toxin chimeric proteins was lower than the parental proteins. Other hybrid proteins were non-toxic.
Masson et al., 1992, “Insecticidal properties of a crystal protein gene product isolated from Bacillus thuringensis subsp. kenyae”, Appl. Environ. Microbiol. 58: 2, 642-646, reported that one of the Cry1 δ-endotoxins, namely Cry1Ea does not exhibit toxicity against Spodoptera larvae. Further, the reference may be made to Bosch et al., 1994, “Recombinant Bacillus thuringensis Crystal protein with new properties: possibilities for resistance management”, Bio/Tech 12: 915-918, wherein many chimeric genes have been developed following in vivo recombination of cry1Ca and cry1Ea genes. The δ-endotoxin expressed from one of the chimeric genes, which consisted of domain I and II of Cry1Ea and domain III of Cry1Ca protein exhibited larvicidal activity. The transfer of domain III of Cry1Ca to Cry1Ea protein gave an insecticidal protein. However, the chimeric toxin was not an improved toxin over the Cry1Ca, which is best-reported toxin to Spodoptera sp. Another chimeric toxin exhibited very poor toxicity. The remaining chimeric toxins were either unstable or non-toxic.
Reference may be cited as Rang et al., 1999, “Interaction between functional domain of Bacillus thuringensis insecticidal crystal protein”, Appl Environ Microbiol, 65, 7: 2918-25, wherein many chimeric genes have been developed by exchanging the regions coding for either domain I or domain III among Cry1Ab, Cry1Ac, Cry1Ca and Cry1Ea δ-endotoxins and checked their stability in E. coli and plasma membrane permeability of Sf9 cells. A chimeric toxin (consisting of domains I and II of Cry1Ca and domain III of Cry1Ab) was more toxic than the parental toxins. Exchange of domain III of Cry1Ab with that of Cry1Ca made the chimeric protein more active than the Cry1Ca protein. Proteins with the exchange of other domains were either unstable or less toxic than the parent proteins. However, the toxicity of the chimeric protein to insect larvae was not tested. Pore formation in insect cell line was compared but that cannot be correlated with the insecticidal activity of the δ-endotoxin.
Reference may be made to Chandra et al., 1999, “Amino acid substitution in alpha-helix 7 of Cry1Ac δ-endotoxin of Bacillus thuringensis leads to enhanced toxicity to Helicoverpa armigera Hubner”, FEBS Lett. 458: 175-179; wherein a hydrophobic motif in the C-terminal end of the fragment B of diphtheria toxin was found to be homologous to helix α7 of δ-endotoxins. Upon substitution of helix α7 of Cry1Ac protein by this polypeptide, the chimeric protein exhibited 7-8 fold enhancement in toxicity towards Helicoverpa armigera. The increased toxicity was due to higher pore forming ability.
These examples establish the potential of protein engineering for the improvement of native toxins, to develop commercially useful δ-endotoxins.
Most of the lepidopteran pests are polyphagous in nature. Spodoptera is a common lepidopteran insect and its 5 species (litura, littoralis, exigua, frugiperda and exempta) are found worldwide. Spodoptera littoralis (the Egyptian cotton leaf worm, CLW) is a major pest of cotton and other crops of agronomical importance in Europe (the reference Mazier et al., 1997, “The cryIC gene from Bacillus thuringensis provides protection against Spodoptera littoralis in young transgenic plants”, Plant Sci. 127: 179-190, may be cited for this). It is a notorious pest of cotton, groundnut, chilli, pulses and several vegetable crops, especially in warm and humid regions, as the southern parts of India. High fecundity, short life cycle, destructive feeding habits and often-reported emergence of resistance to chemical insecticides have made the control of Spodoptera an increasing agricultural problem. Reference may be made to Bai et al., 1993, “Activity of insecticidal proteins and strains of Bacillus thuringensis against Spodoptera exempta (Walker)” J. Inverteb. Pathol. 62: 211-215, wherein it is discussed that the young larvae are susceptible to certain δ-endotoxins, but the larvae beyond 2nd instar display considerable tolerance. This has been attributed to the high content of alkaline proteases in the gut juice (the reference Keller et al., 1996, “Digestion of δ-endotoxin by gut proteases may explain reduced sensitivity of advanced instar larvae of Spodoptera littoralis to CryIC”, Insect Biochem. Mol. Biol. 26: 365-373, may be cites for this).
Four different δ-endotoxins have been reported to cause low level of mortality to the Spodoptera sp. Of these, Cry1Ca is the most effective toxin. The plants expressing Cry1Ca at a high level caused mortality and hence conferred protection against early instar larvae (the references Mazier et al., 1997, “The cryIC gene from Bacillus thuringiensis provides protection against Spodoptera littoralis in young transgenic plants”. Plant Sci. 127: 179-190 and Strizhov et al., 1996, “A synthetic cry1C gene, encoding a Bacillus thuringensis δ-endotoxin, confers Spodoptera resistance in alfalfa and tobacco” Proc. Natl. Acad. Sci. U.S.A. 93: 15012-15017 may be cited for this). However, complete protection against Spodoptera has not been reported in any case. The larvae in advanced developmental stages are not killed at moderate levels of the known δ-endotoxins. Hence, transgenic plants expressing Cry1Ca are not as effective as desirable in protection against Spodoptera. Other genes, like cry1Cb, cry1Ea and cry1F have not been employed for the development of transgenic plants against Spodoptera because of their comparatively low toxicity. Cry1Cb δ-endotoxin is 5-fold less toxic than Cry1Ca. The toxicity of Cry1Ea δ-endotoxin is very low and is disputed in certain reports (Masson et al., 1992 and Bosch et al., 1994 may be cited for this). Cry1F exhibits mild toxicity to Spodoptera larvae (Chambers et al., 1991 may be cited for this).
Reference may be made to Kalman et al., 1993, “Cloning of a novel cryIC-type gene from a strain of Bacillus thuringensis subsp. Galleriae”, Appl. Environ. Microbiol. 59: 4: 1131-1137, wherein Cry1Cb δ-endotoxin is reported to be 5-fold less toxic than Cry1Ca. First two domains of these proteins are highly homologous (92% identical). A major difference is observed in domain III that exhibits only 48% homology. Higher toxicity (5-fold) of Cry1Ca over Cry1Cb δ-endotoxin suggested us that domain III of Cry1Ca might have an important role in its efficacy. The toxicity of Cry1Ea δ-endotoxin is very poor as it binds to the receptor very weakly in the midgut of Spodoptera exigua but the exchange of Domain III of Cry1Ca with Cry1Ea, made the latter toxic. This suggests the role of Domain III of Cry1C protein in receptor binding in the midgut of Spodoptera (the reference Bosch et. al., 1994, “Recombinant Bacillus thuringensis Crystal protein with new properties: possibilities for resistance management”. Bio/Tech 12: 915-918, may be cited for this). In this publication, Bosch et al. (1994) established the advantage of hybrid toxin as it binds to a receptor where Cry1Ca does not bind. However, the toxicity of both, the native Cry1Ca and the hybrid Cry1Ea was comparable. They filed a patent (U.S. Pat. No. 5,736,131) in which 1.9-fold improvement in the toxicity towards Spodoptera exigua was claimed. The difference in results in the publication that reports no enhancement in toxicity and in the patent that claims 1.9 fold improved toxicity, makes the overall picture unclear.
Plant genetic engineering technology has made significant progress during the last 10 years. It has become possible to stably introduce foreign genes into plants. This has provided exciting opportunities for modern agriculture. Derivatives of the Ti-plasmid of the plant pathogen, Agrobacterium tumefaciens, have proven to be efficient and highly versatile vehicles for the introduction of foreign genes into plant tissue. In addition, a variety of methods to deliver DNA, such as electroporation, microinjection, pollen-mediated gene transfer and particle gun technology, have been developed for the same purpose.
The major aim of plant transformation by genetic engineering has been crop improvement. A substantial effort has been made for engineering the plants for useful traits such as insect-resistance. In this respect, progress in engineering insect resistance in transgenic plants has been achieved through the use of genes, encoding δ-endotoxins, from B. thuringiensis strains. Since δ-endotoxins possess a specific insecticidal spectrum and display no toxicity towards other non-host animals and humans, these are highly suited for developing commercially useful plants. No other protein is known which shows as high toxicity as (at ppm levels) and is still as safe to non-target organisms as the δ-endotoxins.
The feasibility of generating insect-resistant transgenic crops expressing δ-endotoxins and their success in commercial agriculture has been demonstrated. (References may be made to Vaeck et al., 1987, “Transgenic plants protected from insect attack”, Nature. 328: 33-37; Fischoffet al., 1987, “Insect tolerant transgenic tomato plants”, Bio/Tech. 5: 807-813”; Barton et al., 1987, “Bacillus thuringensis δ-endotoxin expressed in transgenic Nicotiana tabaccum provides resistance to lepidopteran insects”, Plant Physiol. 85: 1103-1109 may be made for this). Transgenic plants offer an attractive alternative to insect control in agriculture, which is at the same time safe, environment friendly and cost-effective. Successful insect control has been observed under field conditions (Reference may be made to Delannay et al., 1989; Meeusen and Warren, 1989).
A reference may be cited to Von Tersch et al. 1991, “Insecticidal toxins from Bacillus thuringensis subsp kanyae: Gene cloning and characterization and comparison with B. thuringiensis subsp kurstaki Cry1A(c) toxin” in Appl and Environ Microbial, 57: 2: 349-58, wherein two variants of cry1Ac were isolated from two different strains. Their amino acid composition was different at 7 positions. Both the δ-endotoxins were expressed in E. coli and toxicity experiment was conducted. The two toxins did not exhibit any difference in efficacy towards target pests.
In another study (Schnepf et al., 1998, “Bacillus thuringensis and its pesticidal crystal proteins”, 62: 3, 775-806), amino acid residues GYY of Cry1Ac δ-endotoxin at position 312 to 314 were altered to replace these with ASY, GSY and GFS. No difference in toxicity of the three proteins was noticed.
There are two natural variants of Cry1C δ-endotoxin namely Cry1Ca and Cry1Cb. These proteins show 81% (Schnepf et al., 1998, “Bacillus thuringensis and its pesticidal crystal proteins”, 62: 3, 775-806) amino acid sequence identity. Despite this difference, both the toxins are toxic to their target pest, though there is some difference in the level of toxicity. Their host range is also same. (Kalman et al., 1993. “Cloning of a noval cry1C type gene from a strain of Bacillus thuringensis subsp galleriae” Appl. Environ Microbial 59: 4: 1131-37)